crystallization of the isobutylphosphocholine^cholesterol ... · vladimir a. karasev a;*, vladimir...

Crystallization of the isobutylphosphocholine^cholesterolîsobutanol(1:3:3) complex and its investigation by X-ray analysis: interaction of

phopholipid headgroups with cholesterol

Vladimir A. Karasev a;*, Vladimir S. Fundamensky b, Irina I. Bannova c,Valerija D. Franke c, Vassily E. Stefanov c

a St. Petersburg State Electrotechnical University, 197376 St. Petersburg, Russiab Industrial-Research Centre `Burevestnik', 195272 St. Petersburg, Russia

c St. Petersburg State University, 199034 St. Petersburg, Russia

Received 26 October 1999; received in revised form 20 January 2000; accepted 31 January 2000

Abstract

A crystal complex consisting of the isobutyl analog of phosphatidylcholine (PC) (isobutylphosphocholine), cholesterol,and isobutanol with molecular ratio 1:3:3 was obtained and investigated by means of X-ray analysis. The complex wasshown to correspond to the monoclinic system (sp. gr. P21): a = 16.994(10), b = 11.314(7), c = 28.164(15), L= 104.07(3),V = 5252.63 Aî 3, Z = 2, Dcalc = 1.0273 g/cm3. The isobutylphosphocholine molecule is the key component of the complex. Pairsof hydrogen bonds are formed between the ÿNO^PÔNÿ group of the isobutylphosphocholine molecule and CÔH groups oftwo cholesterol and two isobutanol molecules. The third molecules of cholesterol and isobutanol are H-bonded with theÿNO^PÔNÿ group of the isobutylphosphocholine molecule via CÔH groups of isobutanol and cholesterol, respectively. Thecrystal structure is built up by translation of the complex in multiplicate along the two-fold axis in the direction of axis b. Itcontains bands formed by isobutylphosphocholine molecules alternately changing their direction. They are fixed by virtue oftwo zones of electrostatic interactions of the type ÿNO^PÔNÿT�N(CH3)3 and are more or less parallel to the bc plane. Thestructure also contains three-layer domains formed by cholesterol molecules perpendicular to isobutylphosphocholine bands.In the direction of the c-axis isobutylphosphocholine bands alternate with the layers of cholesterol molecules herewithreproducing repeated blocks. The obtained structure is compared with that of crystals of phospholipids and cholesterol andits derivatives. ß 2000 Elsevier Science B.V. All rights reserved.

Keywords: Phospholipid analog; Cholesterol ; Crystal complex; X-ray analysis; Biomembrane modeling

1. Introduction

Cholesterol is one of the most common lipid mol-ecules in the composition of biomembranes of eu-karyotic organisms [1^10]. Its structure contains acyclopentaneperhydrophenanthrene nucleus (CPP-

nucleus), formed by three hexacycles (A, B, C) andone pentacycle (D), and also a side-chain [1]. Choles-terol is alcohol with CÔH-substituent in the thirdposition.

In the biomembrane structure cholesterol is boundwith protein, the intensity of binding being depen-dent on the type of protein participating in suchinteraction [5^7]. Cholesterol was shown to enterinto the composition of detergent-resistant mem-

0005-2736 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved.PII: S 0 0 0 5 - 2 7 3 6 ( 0 0 ) 0 0 1 6 2 - 0

* Corresponding author.

BBAMEM 77830 12-5-00

Biochimica et Biophysica Acta 1466 (2000) 23^38www.elsevier.com/locate/bba

brane domains (DRMS), the latter also containingsphingolipids, including sphingomyelin, and choles-terol-binding proteins [6,7]. Cholesterol may contrib-ute almost 50% to the total lipid content of the bio-membrane. Particularly high is the cholesterolcontent in plasmatic membranes, whereas in othertypes of membranes (endoplasmic reticulum, nucleusmembrane, etc.) it is not that signi¢cant [5]. Withinthe bilayer structure of biomembrane both symmet-rical and asymmetrical distribution of cholesterol inthe outer and inner monolayers may occur [4].

An equimolar ratio of cholesterol and phospholip-ids (PL) is recorded [2,3] in many membranes, e.g. inmyelin membranes. The occurrence of di¡erent typesof molecules implies a possibility of complex forma-tion between them. In fact, such complexes were dis-covered in mixed lipid^cholesterol micelles. Nor-mally, for their investigations pure synthetic PL,e.g. dipalmitoyl PC, and di¡erent sterols, particularlycholesterol, are used [9]. These investigations showedthat in the bilayer lipid membranes the CPP-nucleusand hydrophobic cholesterol side chain are parallelto the PL fatty acid chains. The side chains of chol-esterol molecules are localized in the inner region ofthe bilayer and the C^OH groups of CPP-nuclei arein the vicinity of the ester bonds of PL molecules onthe water^membrane boundary [5,10]. The packingpattern of cholesterol molecules in biomembranes isgenerally assumed as similar to that of cholesterol^PL complexes [6].

The application of the method of electron micros-copy to biomembranes treated with antibiotic ¢lipinallowed to reveal periodic structures - ¢lipin^choles-terol complexes [11]. However, up to now there areno precise data on the packing pattern of either cho-lesterol or its complexes with PL and proteins inbiomembranes.

The structure of the crystals of cholesterol mole-cules and their derivatives as well as of PL moleculesis well investigated [8,12]. However, there are practi-cally no data on the structural studies of cholesterolcomplexes with PL molecules or with their frag-ments. Of particular interest are interactions arisingbetween PL bipolar groups and cholesterol which areassumed in the zone-block model of biomembrane[13^23].

Earlier [23] we investigated the structure of a PCanalog, where the diglyceride radical is replaced by

the isobutyl one (isobutyl-2-(trimethylammonium)-ethylphosphate - IPC), the latter forming a complexwith isobutanol (IB) and water. The obtained struc-ture allowed to presume the formation of a complexbetween IPC and cholesterol. The aim of the presentwork was to obtain IPC complexes with cholesterol,their crystallization, investigation of molecular andcrystal structure.

2. Materials and methods

2.1. Materials

To obtain complex crystals we synthesized isobu-tyl-2-(trimethylammonium)-ethyl phosphate andused cholesterol purchased from Calbiochem. IB (re-agent grade) used for growing crystals was prelimi-narily dried by distillation (the boiling temperatureof the collected fraction was 107.5³C). Earlier [23],complex crystals with IPC were grown from thatsolvent.

2.2. Synthesis of isobutylphosphocholine

IPC was synthesized by the method proposed in[24]. The intermediate cyclic derivative isobutyldi-oxophospholane was obtained from isobutyloxo-phosphodichloride and ethylene glycol. Then thecycle was opened with the simultaneous introductionof a trimethylammonium group. Details of the pro-cedure of the chemical synthesis of the compoundcan be found in [23]. The identi¢cation of IPC wascarried out by NMR-spectroscopy [20].

2.3. Crystallization of the complexes

Joint crystallization of IPC with cholesterol wasperformed by evaporation of the solvent IB. Crystalsformed at di¡erent values of the ratio IPC:cholester-ol were examined. Crystals were observed under themicroscope equipped with an adapter enabling toregulate temperature of the microscopic stage (Nage-ma, Germany). Herewith temperature-dependentchanges in the aggregative state and birefringencewere monitored. Crystals with the properties di¡er-ent from those of IPC and earlier obtained [23] IPCcomplex with IB were discovered. Thus, the new

BBAMEM 77830 12-5-00

V.A. Karasev et al. / Biochimica et Biophysica Acta 1466 (2000) 23^3824

crystals manifested phase transition in the tempera-ture range 75^80³C. Their melting temperature was170³C, which is signi¢cantly di¡erent from that ofIPC (240³C) and cholesterol (149³C).

Samples of IPC (26 mg) and cholesterol (19.4 mg)(molar ratio 2:1) were weighed in 5 ml test tubes(50 mm long, diameter 10 mm). Both componentswere dissolved together in 400 Wl IB heated to 50^60³C. Crystals were grown in a desiccator (capacity0.5^1l) during 7^10 days at room temperature at rel-ative humidity about 12% by a slow evaporation ofthe solvent (above 50^100 ml) oversaturated watersolution of LiCl). Two or three test tubes wereplaced into a desiccator at some angle which led tothe formation of a slanted meniscus. This increasedthe surface of evaporation and enhanced crystal for-mation on the test-tube walls. A small amount ofliquid (about 50 Wl) was left on the bottom of thetest tube. In spite of a two-fold excess of IPC, theratio IPC:cholesterol in the complexes was shiftedtowards the latter, being equal to 1:3.

2.4. Determination of the composition and ratio of thecomponents in the complex crystals

Preliminary information on the composition andratio of the components in crystals is necessary fordetermination of their structure. The loss of masscaused by heating the crystal samples at 50^70³Cto the constant weight was about 15%, which is ap-parently associated with the evaporation of incorpo-rated IB. The phosphorus content in the crystals isapproximately equal to 1.95 þ 0.02%, which testi¢esto the presence of IPC in the crystal structure. The-oretical calculations revealed that the complex crys-tals contain three cholesterol and three IB moleculesper one IPC molecule. These preliminary results werecon¢rmed by the subsequent determination of thecrystal structure.

2.5. X-ray analysis

Crystals for X-ray analysis were selected by thephotographic method. To avoid decomposing, crys-tals were placed in the capillary tubes. Data wereobtained with a monocrystal di¡ractometer SyntexP21 (MoKK radiation, graphite monochromator) inthe range 2^50³ of the angle 2a. In the experiments,

9443 independent re£exes were recorded by the g-method from a monocrystal 7U1U0.2 mm. Forthe subsequent analysis 3159 re£exes meeting thecondition Fhkl s 5.00 cF were taken. Data reductionwas performed by the CSD programs [25] and in-cluded the long-term instability correction and Lp-correction. The determination of the structure by thedirect method and subsequent Fourier syntheses werecarried out by means of SIR 97 [26]. The structurewas re¢ned in the anisotropic approximation fornonhydrogen atoms by the block^fullmatrix method.The blocks, each containing 300 parameters to bere¢ned, were composed manually. For each block,parameters selected for subsequent re¢nement proce-dure exhibited the least correlation coe¤cients. Theposition of hydrogen atoms was determined fromgeometrical considerations (hydrogen atoms of meth-yl radicals were calculated on the basis of the as-sumption of non-shielded conformation). Re¢nementwas undertaken within the model of rigid body. Thewhole procedure was carried out on the basis ofSHELX 97 [27]. A list of re¢ned anisotropic thermalparameters is available from the authors. The ab-sorption correction was introduced by the DIFABSprogram [28]. The structure was re¢ned toRF = 0.0691.

3. Results

3.1. General characterization of the complex andnomenclature

IPC^cholesterol^IB complex was shown to crystal-lize in the monoclinic system (sp. gr. P21) withcell dimensions: a = 16.994(10), b = 11.314(7), c =28.164(15), L= 104.07(3)³, V = 5252.63 Aî 3, Z = 2,Dcalc = 1.0273 g/cm3. There are three cholesterol(Chol 1, Chol 2, Chol 3) and three IBs (IB 1, IB 2,IB 3) molecules per one IPC molecule in the inde-pendent part of the crystal cell. This suggests that thecomplex crystal is solvate. Fig. 1 represents the ar-rangement of molecules in the complex crystal andnumbering of atoms in the structure.

Numbering of atoms in IPC and IB molecules issimilar to that used in [23]. Numbering of atoms incholesterol molecules corresponds to the convention-al nomenclature [1] with the upper subindex desig-

BBAMEM 77830 12-5-00

V.A. Karasev et al. / Biochimica et Biophysica Acta 1466 (2000) 23^38 25

nating the number of the molecule in the crystal. Thecoordinates of atoms and equivalent thermal param-eters (Ueq) are given in Table 1. Geometric parame-ters of the molecules (distances between the atomsand valence angles) are calculated and availablefrom the authors.

3.2. Structure of the molecules making up theIPC^cholesterolîsobutanol (1:3:3) complex

3.2.1. Isobutylphosphocholine moleculeAminoethyl and isobutyl branches are bonded to

the phosphate group of IPC via ester oxygens O3 andO4: Distances PÔ for the ester atoms (1.599 Aî and1.561 Aî ) are signi¢cantly larger than for atoms O1

and O2 (1.454 Aî and 1.471 Aî ). Closeness of the twolatter values testi¢es to the absence of protons asso-ciated with O1 and O2. Similar observations weremade with the IPC and isobutyl-2-aminoethyl phos-phate (isobutylphosphoethanolamineÎPEA) mole-cules investigated earlier [16,21,23].

The torsion angles for the aminoethyl fragment ofIPC in the IPC^cholesterolÎB complex are given inTable 2. For comparison, data on other PL analogsinvestigated earlier, i.e. IPEA^HCl (molecules AP

and A) [21], IPEA [16], IPC^H2O and IPC^H2OÎB [23] are also provided. As seen from Table 2,these angles are most close to those observed in themolecule AP (IPEA^HCl), where atoms P1 and C2

also have a gauche conformation similar to the con-sidered compound. In other molecules, containingthe amino group (IPEA^HCl, molecule A; IPEA),these atoms exhibit trans-conformation, whereas inthe molecules with the choline group the conforma-tion is intermediate. Interatomic distances and va-lence angles in the aminoethyl fragment of the com-plex are also closer to those observed in the moleculeAP [21].

As seen from Fig. 1, the isobutyl branch is almostperpendicular to the aminoethyl one. It was pre-sumed in [21] that the trans-conformation of the ami-noethyl branch is energetically not favored and,hence, is compensated for by high values of thermaloscillations of the isobutyl branch. This results in asubstantial reduction of interatomic distances. Thatis why we expected that, owing to the gauche con-formation of the aminoethyl branch of the IPC mol-ecule of the complex, thermal oscillations and inter-atomic distances in the isobutyl branch wouldassume values usual for similar compounds. Though

Fig. 1. Isobutylphosphocholine^cholesterolÎB complex (1:3:3): conformation and arrangement of molecules; numbering of atoms inthe crystal structure. IPC, isobutylphosphocholine; IB 1ÎB 3, isobutanol molecules; Chol 1^Chol 3, cholesterol molecules. Filledcircles denote carbon atoms. Carbon atoms of more distant cholesterol and IB molecules are represented by hatched circles.

BBAMEM 77830 12-5-00


the distance C3^C4 is slightly larger than similar dis-tances in the molecules A (IPEA^HCl) and IPC,studied earlier [21,23], our assumption about the re-lationship presumably existing between the geometryand dynamics of the isobutyl fragment, on one hand,and the conformation of the aminoethyl fragment,on the other, proved false.

3.2.2. Cholesterol moleculesIn the investigated complex the conformation of

the cycles composing CPP-nuclei of the cholesterolmolecules (Chol 1, Chol 2, Chol 3) is close to thestandard one. Cycles A and C have chair, cycle B,semichair and cycle D, envelope conformation. Theconformation of the cycle B in each molecule is ac-counted for by the presence in the cycle of the doublebond between Ci

5 and Ci6 atoms (upper index i des-

ignates the cholesterol molecule assuming values 1, 2,3). As a consequence, Ci

10, Ci5, Ci

6 and Ci7 lie in one

plane while Ci8 and Ci

9 atoms are situated on theopposite sides of the plane. Angular methyl groupsassociated with Ci

10 and Ci13 atoms are directed to

one side from the plane of the CPP-nucleus.The side chains of cholesterol molecules possess

almost identical conformation which is con¢rmedby the values of torsion angles (Table 3). This tableand Fig. 1 clearly illustrate, that in all three choles-terol molecules the side chain is fully extended. Thevalues of angles between the perpendiculars to theplane passing through the atoms of the D-cycle andthat passing through Ci

17, Ci20, Ci

22, Ci23, Ci

24, and Ci25

atoms of the side chain are close to each other andvary in the range 43.7³^47³. The side chains andCPP-nuclei are extended along one direction.

3.3. Structure of the complex

As seen from Figs. 1 and 2a, free oxygens of thephosphate group (ÿNO1^PÔNÿ

2 ) of the IPC moleculein the independent part of the crystal cell contributeto the formation of hydrogen bonds with cholesteroland IB molecules (Table 4). Thus, the C10Ô5H-group of the IB 1 molecule and the C18Ô7H-groupof the IB 3 molecule form such a bond with the O1

oxygen of the ÿNO1^PÔNÿ2 group. Distances O5Ô1

and O7Ô1 are equal to 2.69(2) Aî and 2.61(2) Aî ,respectively. Angle O5Ô1Ô7 is equal to 92.8(6)³.One of the above atoms (O7) forms a hydrogen

bond with the C33Ô3

1H-group of a cholesterol mole-cule (Chol 3). Distance O7Ô3

1 is equal to 2.65(2) Aî

and the value of angle O31Ô7Ô1 is 109.3(6)³. Oxy-

gen O2 of the phosphate group participates in theformation of two hydrogen bonds with the C1

3Ô1

1H^group of the Chol 1 molecule and C23Ô2

1H-group of the Chol 2 molecule. Distances O2Ô1

1and O2Ô2

1 are equal to 2.67(2) and 2.71(2) Aî , re-spectively, and the value of the angle at O2 is101.8(5)³. Though the planes of the CPP-nuclei ofthe two cholesterol molecules and the side chains(Fig. 1) are approximately parallel, the moleculesare turned to about 180³ with respect to the planeof the ¢gure, so that the angular methyl groups areoriented in the opposite directions. The same is truefor the methyl groups of the C1

21 and C221 side chains.

The C13Ô1

1H-group of the Chol 1 molecule isbonded with the C14Ô6H-group of IB 2 by a hydro-gen bond, distance O6Ô1

1 being equal to 3.03(3) Aî .Thus, atom O1

1 as well as atoms O2 and O7 contrib-ute to two hydrogen bonds leading to the value of105.5(6)³ for the angle O2Ô1

1Ô6. Hence, as seenfrom Fig. 1, two cholesterol molecules (Chol 1 andChol 2) interact directly with the oxygen atom O2 ofthe IPC phosphate group, whereas the third choles-terol molecule (Chol 3) is bound with the IPC mol-ecule via the C18Ô7H-group of the IB 3 molecule.Two IB molecules (IB 1 and IB 3) are directly asso-ciated with the oxygen atom O1 of the IPC phos-phate group. The third molecule (IB 2) is bound toIPC via the C1

3Ô11H-group of the Chol 1 molecule.

Therefore, each free oxygen of the ÿNO1^PÔNÿ2

group of the IPC molecule participates in the forma-tion of two hydrogen bonds.

All three molecules are extended along axis c ofthe cell. Angles between the planes Chol 1^Chol 2,Chol 1^Chol 3, Chol 2^Chol 3 of cholesterol mole-cules assume the following values: 39.4³, 374.8³ and371.1³, respectively. Thus, the planes of Chol 1 andChol 2 are nearly parallel, while the plane of thethird molecule (Chol 3) is almost perpendicular tothe ¢rst two planes.

3.4. Packing of the molecules

As seen in Fig. 2a, oxygen ONÿ2 of the phosphate

group of one IPC molecule from the basal complexof the crystal cell is localized in the proximity of the

BBAMEM 77830 12-5-00


Table 1Coordinates of the atoms and equivalent thermal parameters (Ueq) of the IPC^cholesterol^IB (1:3:3) complex

Atom x/a y/b z/c Ueq

1 2 3 4 5

IPCP1 0.6870(4) 0.0816(6) 0.4969(2) 0.087(2)O1 0.6930(8) 30.0436(14) 0.5089(4) 0.115(4)O2 0.7191(7) 0.1689(12) 0.5355(4) 0.109(4)O3 0.5938(8) 0.1177(10) 0.4770(4) 0.094(4)O4 0.7234(8) 0.0908(15) 0.4512(4) 0.115(4)N1 0.4363(9) 0.0176(14) 0.4989(5) 0.083(4)C1 0.5376(11) 0.0386(21) 0.4462(6) 0.103(7)C2 0.4945(15) 30.0425(22) 0.4767(7) 0.121(8)C3 0.7304(17) 0.2001(28) 0.4285(8) 0.151(10)C4 0.7618(24) 0.1995(32) 0.3855(11) 0.211(16)C5 0.7533(35) 0.0982(35) 0.3546(12) 0.374(40)C6 0.7545(31) 0.3149(34) 0.3627(13) 0.296(26)C7 0.3745(17) 0.0740(26) 0.4600(8) 0.176(11)C8 0.3976(17) 30.0737(28) 0.5218(9) 0.176(12)C9 0.4780(14) 0.1005(21) 0.5388(7) 0.141(9)Molecules of CholesterolChol 1O1

1 0.8642(8) 0.2663(23) 0.5652(5) 0.185(9)C1

1 0.9034(10) 0.3923(20) 0.6930(6) 0.119(8)C1

2 0.8815(15) 0.3899(24) 0.6364(7) 0.151(10)C1

3 0.8865(14) 0.2676(24) 0.6175(7) 0.144(10)C1

4 0.9678(14) 0.2170(21) 0.630(7) 0.144(10)C1

5 0.9890(16) 0.2158(19) 0.6932(7) 0.142(10)C1

6 1.0186(17) 0.1193(23) 0.7178(8) 0.169(13)C1

7 1.0431(17) 0.1078(18) 0.7734(8) 0.169(11)C1

8 1.0545(11) 0.2261(14) 0.7988(6) 0.096(6)C1

9 0.9891(9) 0.3159(14) 0.7732(5) 0.085(6)C1

10 0.9856(9) 0.3329(15) 0.7178(6) 0.092(6)C1

11 0.9963(10) 0.4283(15) 0.8026(5) 0.087(6)C1

12 0.9980(10) 0.4169(16) 0.8563(5) 0.089(6)C1

13 1.0623(9) 0.3277(14) 0.8816(5) 0.071(5)C1

14 1.0499(11) 0.2134(15) 0.8519(6) 0.096(6)C1

15 1.1082(15) 0.1265(17) 0.8853(7) 0.148(9)C1

16 1.0988(14) 0.1605(17) 0.9376(6) 0.127(8)C1

17 1.0525(9) 0.2846(13) 0.9316(5) 0.073(5)C1

18 1.1472(8) 0.3784(18) 0.8859(6) 0.106(7)C1

19 1.0544(12) 0.4129(20) 0.7098(6) 0.125(8)C1

20 1.0832(9) 0.3584(14) 0.9778(5) 0.075(5)C1

21 1.0444(12) 0.4814(14) 0.9738(6) 0.103(6)C1

22 1.0702(10) 0.2946(17) 1.0229(6) 0.094(6)C1

23 1.1070(12) 0.3497(21) 1.0711(6) 0.124(8)C1

24 1.0934(13) 0.2864(19) 1.1149(7) 0.121(7)C1

25 1.1287(23) 0.3221(27) 1.1642(7) 0.233(18)C1

26 1.1150(18) 0.2520(23) 1.2038(7) 0.176(11)C1

27 1.1508(27) 0.4476(26) 1.1770(9) 0.267(23)Chol 2O2

1 0.6363(8) 0.2784(11) 0.5940(3) 0.093(4)C2

1 0.6568(10) 0.1546(14) 0.7208(5) 0.081(5)C2

2 0.6239(10) 0.1662(15) 0.6655(5) 0.079(5)C2

3 0.6640(9) 0.2666(15) 0.6458(5) 0.069(5)

BBAMEM 77830 12-5-00


Table 1 (continued)


1 2 3 4 5

C24 0.6538(10) 0.3785(13) 0.6701(5) 0.067(5)

C25 0.6808(8) 0.3696(13) 0.7257(4) 0.049(4)

C26 0.7315(8) 0.4502(12) 0.7502(5) 0.056(4)

C27 0.7618(9) 0.4509(14) 0.8053(5) 0.071(5)

C28 0.7115(8) 0.3736(11) 0.8304(4) 0.049(4)

C29 0.6948(8) 0.2535(12) 0.8035(4) 0.051(4)

C210 0.6464(8) 0.2687(13) 0.7488(4) 0.056(4)

C211 0.6563(10) 0.1683(14) 0.8335(4) 0.080(5)

C212 0.7004(10) 0.1560(13) 0.8873(5) 0.077(5)

C213 0.7091(7) 0.2777(13) 0.9126(4) 0.058(4)

C214 0.7549(8) 0.3543(12) 0.8834(4) 0.051(4)

C215 0.7820(9) 0.4624(12) 0.9158(4) 0.065(4)

C216 0.7979(9) 0.4133(12) 0.9688(5) 0.068(5)

C217 0.7674(8) 0.2811(12) 0.9642(4) 0.060(4)

C218 0.6279(8) 0.3314(16) 0.9148(5) 0.081(5)

C219 0.5572(8) 0.2878(18) 0.7456(6) 0.097(6)

C220 0.7399(9) 0.2435(13) 1.0097(4) 0.062(4)

C221 0.7081(11) 0.1173(17) 1.0046(6) 0.109(8)

C222 0.8045(8) 0.2606(16) 1.0570(4) 0.068(4)

C223 0.7791(9) 0.2451(19) 1.1034(5) 0.090(6)

C224 0.8405(10) 0.2641(19) 1.1498(5) 0.093(6)

C225 0.8224(13) 0.2389(27) 1.1975(6) 0.154(12)

C226 0.7518(16) 0.3028(45) 1.2030(7) 0.304(32)

C227 0.8920(13) 0.2599(27) 1.2410(6) 0.170(11)

Chol 3O3

1 0.5648(8) 30.1785(13) 0.6106(4) 0.115(4)C3

1 0.6222(11) 30.2666(18) 0.7431(6) 0.106(7)C3

2 0.6104(12) 30.2757(15) 0.6882(6) 0.100(6)C3

3 0.5716(11) 30.1672(16) 0.6620(5) 0.088(6)C3

4 0.4929(10) 30.1468(19) 0.6724(6) 0.098(6)C3

5 0.4996(10) 30.1464(16) 0.7268(7) 0.087(6)C3

6 0.4631(10) 30.0607(17) 0.7455(6) 0.090(6)C3

7 0.4603(10) 30.0466(15) 0.7986(6) 0.094(6)C3

8 0.4909(9) 30.1541(15) 0.8295(5) 0.077(5)C3

9 0.5651(9) 30.2075(14) 0.8133(5) 0.080(5)C3

10 0.5424(10) 30.2451(14) 0.7582(5) 0.074(5)C3

11 0.6069(11) 30.3047(17) 0.8478(6) 0.102(6)C3

12 0.6264(9) 30.2726(16) 0.9018(6) 0.093(6)C3

13 0.5500(8) 30.2298(13) 0.9178(5) 0.067(5)C3

14 0.5141(9) 30.1271(13) 0.8834(5) 0.073(5)C3

15 0.4520(10) 30.0697(17) 0.9074(7) 0.102(6)C3

16 0.4909(10) 30.0892(15) 0.9635(6) 0.092(6)C3

17 0.5655(8) 30.1731(12) 0.9681(5) 0.066(5)C3

18 0.4871(10) 30.3294(15) 0.9140(6) 0.094(6)C3

19 0.4891(13) 30.3537(16) 0.7482(7) 0.126(8)C3

20 0.5816(9) 30.2456(14) 1.0155(5) 0.070(5)C3

21 0.6509(10) 30.3319(15) 1.0166(6) 0.094(6)C3

22 0.6009(10) 30.1677(15) 1.0603(6) 0.086(6)C3

23 0.6116(10) 30.2243(17) 1.1090(6) 0.092(6)C3

24 0.6306(12) 30.1388(22) 1.1512(7) 0.137(9)C3

25 0.6490(17) 30.1860(25) 1.2006(8) 0.165(12)C3

26 0.7029(18) 30.2836(23) 1.2116(9) 0.175(11)

BBAMEM 77830 12-5-00


nitrogen atom of the N�1 (CH3)3-group of anotherIPC molecule related to the ¢rst one by rotationabout the screw axis 21. The distance between theabove atoms is equal to 4.71(2) Aî (Table 4). Subse-quent translation of the two molecules along axis bof the unit cell results in the formation of a contin-uous chain of IPC molecules with aminoethyl frag-ments having antiparallel orientation. The aminoeth-yl fragments lie in a plane approximately parallel toplane ab, whereas isobutyl fragments are roughlyperpendicular to that plane. Since IPC moleculesare interrelated by a double screw axis and two ofthem are present in the unit cell, two lines of isobutyl

fragments parallel to axis b emerge. The isobutylfragments have a di¡erent orientation with respectto the plane of aminoethyl fragments.

Another free oxygen atom (ÿNO1) of the phosphategroup from the IPC molecule, related to the basalone by the axis 21 (Fig. 2a), is associated with thenitrogen atom of the trimethylammonium group ofthe basal molecule. The distance between these atoms(5.41(2) Aî ) is somewhat larger than the distance O2^N1. The presence of the opposite charges of thegroups ÿNO1^PÔNÿ

2 and N�1 (CH3)3 together withthe absence of shielding groups allow to interprettheir interaction as electrostatic or ionic. Thus, two

Table 2Comparison of the torsion angles in the of PL analogs

Torsion angles IPC^CholesterolÎB IPEA^HCl mol.AP IPEA^HCl mol.A IPEA IPC^H2O IPC^H2OÎB

C6^C4^C3Ô4 3171.9³ 170.3³ 175.5³ 180.0³ 46.6³ -69.0³C4^C3Ô4^P1 177.7³ 3179.3³ 3159.6³ 3143.4³ 150.0³ 3179.5³C3Ô4^P1Ô3 66.5³ 53.0³ 62.8³ 62.3³ 76.4³ 373.3³O4^P1Ô3^C1 373.0³ 62.9³ 58.3³ 73.2³ 60.5³ 359.9³P1Ô3^C1^C2 91.8³ 3100.5³ 3176.9³ 3157.0³ 3131.5³ 131.9³O3^C1^C2^N1 70.4³ 67.7³ 62.6³ 59.2³ 72.0³ 379.1³C1^C2^N1^C8 3175.1³ 163.3³ 348.0³ 42.8³ 3178.0³ 177.5³

Table 1 (continued)


1 2 3 4 5

C327 0.6675(17) 30.0935(24) 1.2408(8) 0.193(14)

Molecules of IBIB 1O5 0.6845(14) 30.2395(21) 0.4540(7) 0.221(10)C10 0.7520(14) 30.2566(34) 0.4336(11) 0.211(17)C11 0.7328(15) 30.2841(44) 0.3862(10) 0.239(24)C12 0.6563(18) 30.3252(53) 0.3609(11) 0.361(42)C13 0.7923(11) 30.2980(24) 0.3625(6) 0.274(24)IB 2O6 0.8622(11) 0.4847(24) 0.4979(6) 0.398(29)C14 0.9213(11) 0.4645(24) 0.5225(6) 0.790(132)C15 0.9989(11) 0.4928(24) 0.4949(6) 0.665(115)C16 1.0764(11) 0.4791(24) 0.5105(6) 0.464(58)C17 0.9887(11) 0.5745(24) 0.4639(6) 0.730(136)IB 3O7 0.7081(10) 30.1645(22) 0.5894(7) 0.201(10)C18 0.7770(17) 30.2352(28) 0.6079(15) 0.326(32)C19 0.8260(17) 30.1448(30) 0.6317(13) 0.346(42)C20 0.8150(36) 30.0859(62) 0.6736(24) 0.630(89)C21 0.8939(16) 30.2051(40) 0.6417(13) 0.327(30)

Note: The atomic fractional coordinates in the unit cell with the error indicated in parentheses.

BBAMEM 77830 12-5-00


Fig. 2. Projection of the crystal structure isobutylphosphocholine^cholesterolÎB complex (1:3:3) on the plane ab. (a) Cross-sectionalong axis c from 0.35^0.65; hydrogen bonds - dotted lines and ionic (electrostatic) bonds - dashed lines. Only CÔ of cholesterolmolecules are shown. Filled circles denote carbon atoms. Carbon atoms of IB situated behind the line of isobutylphosphocholine mol-ecules are represented by hatched circles. The complex from the independent part of the cell is encircled with a dashed line. (b) Pack-ing of cholesterol molecules: cross-section (0.5^1.0) along axis c. Dashed and blank ellipses correspond to cholesterol molecules turnedtowards the line of isobutylphosphocholine molecules with CÔH-groups and side chains, respectively.

BBAMEM 77830 12-5-00


systems of ionic interactions can be distinguished inthe plane of amino phosphate fragments, which pro-vides for the association of IPC molecules along theb-axis (Fig. 2a,b, dotted lines).

Cholesterol molecules are connected with the bi-layer via the above described system of hydrogenbonds (see Section 3.3). As seen in Fig. 3, all choles-terol molecules are roughly perpendicular to theplane of the bilayer. The axis of the cholesterolmolecule passing through the aliphatic side chain

and planes of the cycles is almost parallel to thec-axis.

All three cholesterol molecules from the basalcomplex of the crystal cell associated with one IPCmolecule have the side chains oriented downwardswith respect to the plane of the ¢gure (Fig. 2b).Two of them (Chol 2 and Chol 3) are situated onapproximately the same level in bc plane, whereas thethird molecule (Chol 1) lies on a di¡erent level. Thescrew axis 21, having the direction [010] and passing

Fig. 3. Projection of the crystal complex isobutylphosphocholine^cholesterol^IB (1:3:3) on the plane bc. (a) Layers of cholesterol mol-ecules localized along the axis a from 0.45^0.75 of the unit cell. In the left part, the upper layer of cholesterol molecules is removedto show the layer situated below (carbon atoms are represented by hatched circles). (b) The layer of cholesterol molecules localizedalong the axis a from 0.90^1.10 of the unit cell. Periodically repeated blocks are marked with arrows.

BBAMEM 77830 12-5-00


through the point 1/2, 0, 1/2 multiplies the basalcomplex turning the orientation of the cholesterolside chains to the opposite direction from the bilayerplane (non-hatched ellipses).

Thus, three layers of cholesterol molecules perpen-dicular to ab- and parallel to bc- plane emerge in thecell. The ¢rst two layers (Fig. 2b layers I and II)consist of the molecules possessing the same orienta-tion within each particular layer, the orientation ofthe molecules belonging to the two layers being op-posite. This can be better seen from Fig. 3a showinga cross-section (0.25^0.45) of the crystal cell perpen-dicular to axis a and passing through the layer ofcholesterol molecules with the same orientation ofthe side chains. The third layer contains cholesterolmolecules with alternating orientation (Fig. 3b, thecross-section 0.95^1.05). In this layer one cholesterolmolecule belongs to the basal complex and the otherto the complex from the cell situated below. Whilethe ¢rst two layers contain IPC molecules, the latterare absent from the third layer.

Systems of hydrogen bonds surrounding IPC mol-ecules from all sides are insulated by the regions ofVan der Waals' interactions and ionic groups (Fig.2a,b). This is essentially di¡erent from the situationobserved in crystals of IPEA and IPC [16,21,23],where systems of hydrogen bonds unite moleculesof PL analogs into continuous two-dimensionalstructures.

As seen from Fig. 3a,b, the cholesterol moleculesof both cross-sections from the adjacent cells pene-trate each other for a distance equal to the length of

the side chains, whereas the regions of the CPP-nu-clei do not overlap. Contacts between the side chainsand cyclic fragments of the cholesterol moleculesfrom the cells neighboring in the plane ab take placedue to Van der Waals' interactions. These interac-tions also contribute to the contacts between the cellsneighboring in the plane bc. In the layers I and IIwith the same orientation of cholesterol moleculesonly axes of these molecules are parallel, whereasplanes passing through the cyclic fragments of themolecules are not. On the contrary, the angle be-tween the perpendiculars to the planes of the CPP-nuclei in the layer III with the alternating orientationof the molecular axes is about 180³, i.e. the moleculesare more or less parallel. However, their angularmethyl groups have opposite directions. The four-residue fragment with two angular CH3-groups ofthe side chain is complementary to the ¢ve-residuecycle of the adjacent cholesterol molecule. Therefore,the parameter c of the unit cell is determined bythickness of the bilayer and doubled length of theCPP-nucleus.

As seen from Figs. 1 and 3a, IB molecules sur-round IPC molecules from three sides, being local-ized in the lower part of the bilayer and forming akind of solvate medium. The IB 1 molecule isroughly parallel to the isobutyl branch of IPC,

Table 4Hydrogen bonds and ionic interactions in the crystal of theIPC^cholesterol^IB (1:3:3) complex

Symmetry operatora Atoms Interatomicdistances, Aî

Hydrogen bondsO1TO5 2.69(2)O3

1TO7 2.65(2)O1TO7 2.61(2)O2TO2

1 2.71(2)O1

1TO2 2.67(2)O1

1TO6 3.03(3)Ionic interactions[2 1 31 1] ONÿ

2 T�N1 4.71(2)[2 1 0 1] ONÿ

1 T�N1 5.41(2)

The operator: 2: 3x, 1/2+ y, 3zaThe numbers in square brackets show that the ionic bond con-tains an atom the coordinates of which are obtained from theinitial ones (Table 2) by transforming the symmetry operator(¢rst digit) and subsequent translation along axes a, b, and c(respectively, the second, third and fourth digits).

Table 3Torsion angles for side chains of cholesterol molecules

Torsion angles Values of torsion angles

Chol 1 Chol 2 Chol 3

C13^C17^C20^C21 56.4 52.5 52.5C13^C17^C20^C22 3178.2 3176.9 3177.2C15^C16^C17^C20 150.3 153.3 154.4C16^C17^C20^C21 3179.4 3174.7 3174.2C16^C17^C20^C22 55.3 54.7 61.2C17^C20^C22^C23 3170.4 3167.9 3176.1C20^C22^C23^C24 3178.6 3178.5 3178.4C21^C20^C22^C23 62.3 61.4 59.6C22^C23^C24^C25 3177.4 3178.6 3177.3C23^C24^C25^C26 13.3 23.3 45.5C23^C24^C25^C27 3177.0 3175.0 3173.7

BBAMEM 77830 12-5-00


whereas IB 2 and IB 3 molecules have the sameorientation as the aminoethyl branch of IPC. Thesemolecules play an important role in the formation ofthe systems of hydrogen bonds generating the molec-ular complex, though producing no essential e¡ecton the packing of such complexes.

4. Discussion

Three aspects of the presented investigation will bediscussed below: packing of isobutylphosphocholinemolecules, packing of cholesterol molecules; role ofIB molecules. Properties of the investigated complexwill be compared with: the packing pattern of bipo-lar headgroups in the crystals of PL molecules; thestructure of cholesterol crystals and its derivatives;solvate crystals. Other aspects, particularly those re-lated to the biomembrane structure will be dealt within subsequent works.

4.1. Packing of isobutylphosphocholine molecules

Above (Section 3.4.) it was shown that negativelycharged groups ÿNO1^PÔNÿ

2 of IPC molecules inter-act with positively charged groups N�1 (CH3)3 ofneighboring IPC molecules which have the opposite(antiparallel) orientation. Isobutyl branches of thesemolecules are situated on the opposite sides of aband composed by phosphocholine groups. Hence,two parallel zones emerge (Scheme 1, bold; R, iso-butyl residue) due to ionic interactions. DistancesN�^ÿNO are equal to 4.71 Aî and 5.41 Aî which israther long for ionic bonds. Normally, the lengthof such bonds is in the range 3.7^4.5 Aî [12,23].

Apart from ionic interactions there are also otherfactors contributing to the stability of the abovestructure in the investigated crystal. Thus, due tohydrogen bonds between CÔH-groups of Chol 1and Chol 2 molecules and O2 atom of IPC phosphategroup, bipolar heads are retained in the perpendicu-lar position with respect to cholesterol. The packingpattern of cholesterol molecules consisting in stack-ing and side-by-side Van-der-Waals' interactions isalso important for stabilization of the structure.

Similar interdigitating and antiparallel arrange-ment of phosphocholine groups from adjacent mono-layers was observed in the crystals of 3-octadecyl-2-methyl-D-glycero-1-phosphocholine monohydrate(OMPC) [29]. In the crystals of 3-palmitoyl-D-glyc-ero-1-phosphocholine monohydrate (PPC) and 3-hexadecyl-D-glycero-1-phosphocholine chloroformsolvate (HPC) [12] such a packing pattern was shownonly for the dimers of the molecules which belong tothe adjacent monolayers.

In the crystals of 2,3-dimyristoyl-D-glycero-1-phos-phocholine dihydrate (DMPC) [30,31], 2-deoxy-3-lauroyl-glycero-1-phosphocholine monohydrate (de-oxyLPC) [30,32], IPC monohydrate and complex ofIPC monohydrate with IB [23] an essentially di¡erentpacking pattern of bipolar groups takes place. Thus,in DMPC crystals ionic bonds were shown to emergebetween ÿNO^PÔNÿ- and N�(CH3)3-groups of theneighboring molecules belonging to one monolayer.In addition, ÿNO^PÔNÿ-groups of DMPC are H-bonded with each other within one monolayer viawater molecules. Close to each other zig-zag ribbonsof hydration water molecules were observed in themonolayers of molecules of deoxyLPC [30,32], IPCmonohydrate and in the complex of IPC monohy-drate with IB [23]. In the above crystals interactionof headgroup dipoles is observed both within themonolayer and bilayer with the inward orientationof the bipolar groups. Interaction of antiparallelphosphocholine groups of pairs of molecules fromthe adjacent monolayers accounts for the formationof the bilayer.

Zones of interdigitating headgroups were discov-ered not only in PC crystals but also in the crys-tals of dimethyl- and monomethyl-substituted phos-phatidylethanolamine (PE). Such zones contain ionicand hydrogen bonds, e.g. in the crystals of 2,3-dilauroyl-DL-glycero-1-phospho-N,N-dimethylamineScheme 1.

BBAMEM 77830 12-5-00


(DLPEM2) [33] systems, shown in Scheme 2 (R, 2,3-dilauroyl-DL-glycerol radical). In the zones (Scheme2, bold) alternating dipole pairs form hydrogenbonds of the type C^N�HTÿNO^PÔNÿ and ionicbonds, similar to those in the complex analyzed inour work. In the crystals of 2,3-dilauroyl-DL-glycero-1-phospho-N-monomethylamine (DLPEM1) [12]quasi one-dimensional systems of hydrogen bondsformed by ÿNO^PÔNÿ- and C^N�H-groups of bipo-lar heads (Scheme 3; R, 2,3-dilauroyl-DL-glycerolradical) can be observed. Apparently, the formationof the above shown systems of hydrogen bonds isconnected with the replacement of a methyl groupin DLPEM2 by the hydrogen atom. Under certainconditions, pairs of two-dimensional layers contain-ing systems of alternating C^N- and O^PÔ-groupswill probably be discovered in PEA crystals, wherethe nitrogen atom retains three hydrogen atoms.

Formation of the double zones of ionic or hydro-gen bonds from interdigitating antiparallel bipolarheads in PL crystals and in the investigated structureis not related to biomembranes on the conventionalbasis [34]. However, from the view-point of the zone-block model [13^23] such zones situated in the cen-tral region of the biomembrane structure provide for

the lateral transfer of charge (signal) between theblocks of protein, thus playing an important role inthe intramembrane communication [15,19].

4.2. Packing pattern of cholesterol molecules

The main peculiarity in the structural organizationof cholesterol in the investigated structures suggeststhat cholesterol, IPC and IB molecules make up acomplex. The base fragment consists of one IPC mol-ecule, three cholesterol molecules and three IB mol-ecules (Fig. 1). As was shown above (3.3), two cho-lesterol molecules (Chol 1 and Chol 2) formhydrogen bonds directly with the O2-atom of thephosphate group of IPC, whereas the third molecule(Chol 3) is connected with the O1-atom of the IPCmolecule via the CÔH-group of IB 3. MoleculesChol 1 and Chol 2 directly bonded to IPC are situ-ated practically one above the other and their planesare almost parallel. The mutual orientation of theangular methyl groups of these molecules is almostopposite (Fig. 1). Molecule Chol 3, like Chol 1 andChol 2, has a CÔH-group directed towards IPC.The angle between the plane of the CCP-nucleus ofChol 3 and that of the two other Chol molecules isover 70³.

IPC molecules together with IB molecules associ-ated with them form a continuous band. Cholesterolmolecules are bound to it and their main axes areperpendicular to the band (Figs. 1 and 3). Thesemolecules form layers with varying orientation with-in each layer (Fig. 2b and 3).

Prior to the comparative analysis of the investi-gated complex and crystals of cholesterol moleculesand its derivatives let us underline some structuralpeculiarities of this compound. Cholesterol moleculeshave a very conservative CCP-nucleus, which is prac-tically invariant in all previously investigated crystalsof cholesterol and its derivatives [8]. On the contrary,the aliphatic fragment of the molecule is very labileand often it is di¤cult to ¢x it due to a big amplitudeof temperature oscillations. As a rule, a reliable ¢x-ation of these atoms can be achieved at low temper-ature [35]. Therefore, it is clear why the little discrep-ancy we observed in the values of distances andangles of the CCP-nuclei of the investigated structureis within the accuracy of the determination of thepositions of atoms. This also refers to the di¡erenceScheme 3.

Scheme 2.

BBAMEM 77830 12-5-00


between the investigated structure and the structuresof other compounds containing cholesterol and itsderivatives.

Let us compare packing patterns of cholesterolmolecules in di¡erent compounds. It should be notedthat formation of crystals with several molecules inthe independent part of the lattice cell is typical forcholesterol and its derivatives, e.g. anhydrous choles-terol [36], cholesterol monohydrate [37] and othersubstances [38] contain eight independent moleculeseach in the cell and cholesterol hemiethanolate [39]has even twice this number. The number of choles-terol molecules in the independent part of the latticecell of all structures known to us is even. However, inthe cell of the structure investigated by us this num-ber is equal to three. Together with the occurrence ofother bulky molecules in the investigated structure,this accounts for a peculiar packing pattern.

Due to the fact that the number of molecules iseven, it is always possible to distinguish a pair ofindependent molecules which fully determine thepacking pattern in the structure and may explainits pseudosymmetry. Thus, in the structure of choles-teryl acetate [35] there are two independent choles-terol molecules. They have antiparallel arrangementon the adjacent screw axes and lie approximately inone plane perpendicular to the binary screw axis.This results in a stack packing of the molecules.The stacks of molecules form parallel continuouslayers. In such an arrangement the tail fragmentsof one molecule are directed towards the acetyl rad-icals of a molecule from the neighboring stack andlying approximately in one plane perpendicular tothe screw axis. In the next plane the tail fragmentof one molecule and the acetyl radical from theneighboring stack are antiparallel to those consideredabove. Such a pattern was observed in cholesteryliodide [40].

CCP-nuclei of cholesterol can be situated in paral-lel planes or form di¡erent angles with each other.Several types of packing can be obtained with cho-lesterol molecules whose CCP-nuclei lie in the planesapproximately parallel to each other. Stacking maytake place with the cholesterol molecules which areparallel to each other. Stacks can form layers, whereadjacent cholesterol molecules are situated at thesame height. The stacks involved in the formationof such a structure may have two types of mutual

orientation. In the ¢rst case the tail of one moleculeis directed towards hydroxyl of the other. In thesecond case the arrangement pattern is alternating,i.e. in one pair of the adjacent stacks it is tail to tailwhereas in the other it is hydroxyl to hydroxyl. Twoneighboring stacks can be shifted relative to eachother in height. In this case interpenetration of stacksand overlapping of a part of one cholesterol moleculewith another molecule can be observed in the projec-tion of the structure on the CCP-nucleus plane. Theantiparallel arrangement of molecules mentionedearlier [35] can be regarded as the realization of com-plete interpenetration of adjacent stacks. Theoreti-cally other types of packing of cholesterol moleculescan also be conceived [8].

Another pattern suggests that the main element ofthe crystal structure is formed by a pair of moleculeswhich have CCP-nucleus planes practically perpen-dicular to each other. This pattern is obtained withsuch compounds as laurate, nonanoate and dec-anoate of cholesterol [41^43]. The arrangement ofmolecules in their crystals is such that the CCP-nu-cleus of one molecule is situated just opposite theester radical of the other molecule. This fragment isfurther repeated via two-fold screw axes.

In the compound investigated by us, the structuralfragment, consisting of three cholesterol molecules(see above), possesses many common features withboth structures composed by parallel pairs and per-pendicular pairs of cholesterol molecules. Thus,layers I and II in the crystal (Fig. 2b and 3a) consistof cholesterol molecules with the parallel orientation.However, the layers themselves are oriented in theopposite direction, so that pairs of molecules situatedone above the other (Chol 2 and Chol 3) have anti-parallel orientation forming nearly a direct angle. Inthe layer III, molecules Chol 1 have antiparallel ori-entation. In the stacks of molecules from the layer II,the parallel orientation of molecules Chol 1 and Chol2 alternates with the antiparallel arrangement (theangle equal to 70³) of molecules Chol 1 and Chol 3.

Due to the occurrence of a continuous layer con-sisting of phosphocholine residues, approximatelyperpendicular to the axes of cholesterol molecules,the repeated structural blocks in the investigatedcrystal are divided into two regions containing IPCmolecules and cholesterol molecules with interpene-trating tail fragments. This structure resembles that

BBAMEM 77830 12-5-00


of cholesteryl myristate [44] which also reveals twodistinct regions. However, in the latter case myristylradicals interdigitate, whereas in the structure we in-vestigated, this zone is ¢lled with IPC and IB mole-cules.

4.3. The role of IB molecules

IB molecules undoubtedly play an essential role inthe structural organization of the investigated crystalcomplex. Thus, evaporation of IB from the crystalresults in turbidity and amorphization of the latter.As can be seen from Fig. 3, IB and IPC moleculesare localized in the same region. In the biostructures,the side chain of the amino acid serine can be re-garded as an analog of the IB molecule [23]. In thestructure of the complex, IB plays a role which iscomplementary to that of cholesterol, i.e. IB mole-cules ¢ll in the space inaccessible for cholesterol mol-ecules. Besides, IB contributes to the formation of anantisymmetric system of hydrogen bonds (Scheme 4).

As follows from Scheme 4, CÔH-groups of IB 1and IB 3 molecules are bonded to O1 of the phos-phate group, whereas the third CÔH-group, incor-porated in this system, belongs to the Chol 3 mole-cule. CÔH-groups of Chol 1 and Chol 2 moleculesare bonded to the O2 atom, whereas the third CÔH-group of this system belongs to IB 2. Therefore, thetwo systems are related to each other by transitionIB6CChol.

It is worth mentioning that incorporation of sol-vent was observed both in some crystals of PLs, e.g.HPC [12], and cholesterol (cholesteryl hemiethano-late) [39]. In this connection, crystals of the complex

of IPC monohydrate with IB, we investigated earlier[23], are of particular interest. Schematically elementsof this complex are shown in Scheme 5 (R1 : cholineradicals, R2 : isobutyl radicals in IPC, R2 : isobutylradicals in IB). It is seen in the scheme that phos-phate groups and water molecules form a system ofhydrogen bonds. In the lower part of the scheme, IBmolecules (bold) are `suspended' from water mole-cules by means of hydrogen bonds. They wedge inbetween isobutyl radicals of IPC. Just on the basis ofthis structure we obtained the complex of IPC withcholesterol and IB.

Thus, in the present article crystals of the complexof IPC with cholesterol and IB were obtained andtheir molecular and crystal structure was studied. Itwas shown that in the complex IPC molecules arepacked into continuous bands formed by antiparallelinterdigitating bipolar heads. They generate doublezones of C^N�(CH3)3 and ÿNO^PÔNÿ-groups. Thestructure also contains three-layer domains formedby cholesterol molecules perpendicular to isobutyl-phosphocholine bands. Since the packing pattern ofbipolar groups of IPC is very much similar to thatshown for PL molecules in crystals, one should notexclude that in the future some of its elements, par-ticularly zones of bipolar headgroups of PLs, can bediscovered in biomembranes.

By and large, the developed approach opens newprospects in the study of complexes of PL analogswith cholesterol and its derivatives, e.g. creatingcomplexes with plant sterols, steroid hormones, in-corporating cholesterol-binding proteins or theirfragments and so on.

Acknowledgements

We are grateful to Dr. V.V. Luchinin for helpfuldiscussion. A part of this work was supported by the

Scheme 5.

Scheme 4.

BBAMEM 77830 12-5-00


research Grant from Russian Foundation for BasicResearch (code 99-04-49836).

References

[1] L.F. Fieser, M. Fieser, Steroids, Reinolds, New York, Chap-man and Hall, London, 1959.

[2] R.A. Demel, B. de Krui¡, Biochim. Biophys. Acta 457(1976) 109^132.

[3] E.M. Kreps, Lipidy kletochnych membran. Evolutcia lipidovmozga. Adaptatcionnaja functcija lipidov (Lipids of cellularmembranes. Brain lipids evolution. Adaptational function oflipids), Nauka Science Publiushers, Leningrad, 1981, in Rus-sian.

[4] G. Benga, R.P. Holmes, Progr. Biophys. Mol. Biol. 43(1984) 195^257.

[5] P.L. Yeagle, Biochim. Biophys. Acta 822 (1985) 267^287.[6] K. Simons, E. Ikonen, Nature 387 (1997) 569^572.[7] R.J. Schroeder, S.N. Ahmed, Y. Zhu, E. London, D.A.

Brown, J. Biol. Chem. 273 (1998) 1150^1157.[8] B.M. Craven, in: D.J. Hanahan (Ed.), Handbook of Lipid

Research, vol.4, The Physical Chemistry of Lipids, PlenumPress, New York, 1988, pp. 149^182.

[9] J.A. Urbina, S. Pekerar, H.-b. Le, J. Patterson, B. Montez,E. Old¢eld, Biochim. Biophys. Acta 1238 (1995) 163^176.

[10] F.T. Presti, in: R.C. Aloia, J.M. Boggs (Eds.) MembraneFluidity in Biology, vol.4, Cellular Aspects, Academic Press,Orlando, FL, 1985, pp. 97^147.

[11] L. Luciano, E. Reale, H. Konitz, U. Boseck, S. Boseck,J. Histochem. Cytochem. 37 (1989) 1421^1425.

[12] I. Pascher, M. Lundmark, P.-G. Nyholm, S. Sundell, Bio-chim. Biophys. Acta 1113 (1992) 339^373.

[13] V.A. Karasev, Viestnik Leningr. Univers. 9 (1974) 74^86.[14] V.A. Karasev, Zonno-blochnaja model structury biologiche-

skikh membran (Zone-block model of biological membranesstructure), deposited in VINITI, Moscow, 1988, 29 pp., no.5306-V88, in Russian.

[15] V.A. Karasev, V.E. Stefanov, B.I. Kurganov Nadmolecular-nie structury: Organizatzia, Functzionirovanie, Proiskhozh-denie (Supramolecular structures: organization, functioning,origin), in: Itogi nauki i tekhniki, Ser. Biologich. Khimia(Progress in Science and Techniques, Ser. Biolog. Chem.),vol.31, WINITI, Moscow, 1989, 199 pp., in Russian.

[16] V.S. Fundamensky, V.A. Karasev, V.V. Luchinin, Biol.Membr. 9 (1992) 789^802.

[17] V.A. Karasev, V.E. Stefanov, J. Biochem. Organ. 1 (1992)71^87.

[18] V.A. Karasev, V.E. Stefanov, Usp. Biol. Khim. (Adv. Biol.Chem.) 32 (1992) 114^145.

[19] V.A. Karasev, V.V. Luchunin, V.A. Stefanov, Adv. Mater.Opt. Electron. 4 (1994) 203^218.

[20] V.A. Karasev, N.A. Korovnikova, V.A. Gindin, V.E. Stefa-nov, Colloids Surf. A Physicochem. Engin. Aspects 115(1996) 83^87.

[21] V.S. Fundamensky, I.I. Bannova, V.A. Karasev, V.P. Mir-oshkin, V.V. Luchinin, Membr. Cell Biol. 9 (1996) 547^564.

[22] V.A. Karasev, N.A. Korovnikova, V.P. Miroshkin, V.V.Luchinin, V.S. Fundamensky, I.I. Bannova, V.E. Stefanov,Adv. Mater. Opt. Electron. 6 (1996) 1^14.

[23] V.S. Fundamensky, I.I. Bannova, V.D. Franke, V.A. Kara-sev, N.A. Korovnikova, V.V. Luchinin, Membr. Cell Biol.11 (1997) 137^156.

[24] R.L. Magolda, P.R. Jonson, Tetrahedron Lett. 26 (1985)1167^1170.

[25] L.G. Axelrud, U.N. Grin, P.U. Zavalii, V.K. Pecharsky,V.S. Fundamensky, Abstr. of II Eur. Crystallog. Meeting,vol.3, Moscow, 1989, p. 155.

[26] G. Cascarano, A. Altomare, C. Giacovazzo, A. Guagliardi,A.A. Moliterni, D. Siligi, M.C. Burla, G. Polidori, M. Ca-malli, Acta Crystallogr. A52 (Suppl.) (1996) C79.

[27] G.M. Sheldrick, SHELX 97, Manual. University of Go«ttin-gen, Go«ttingen, 1997.

[28] N. Walker, D. Steward, Acta Crystallogr. 39 (1983) 158^165.[29] I. Pascher, S. Sundell, H. Eibl, K. Karlos, Chem. Phys. Lip-

ids 39 (1986) 53^64.[30] H. Hauser, I. Pascher, R.H. Pearson, S. Sundell, Biochim.

Biophys. Acta 650 (1981) 21^51.[31] R.H. Pearson, I. Pascher, Nature 281 (1979) 499^501.[32] H. Hauser, I. Pasher, S. Sundell, J. Mol. Biol. 137 (1980)

249^264.[33] I. Pascher, S. Sundell, Biochim. Biophys. Acta 855 (1986)

68^78.[34] S.J. Singer, G.L. Nicolson, Science 175 (1972) 720^731.[35] P. Sawzik, B.M. Craven, Acta Crystallogr. B35 (1979) 895^

901.[36] H.S. Shich, L.G. Hoard, C.E. Nordman, Nature 267 (1977)

287^289.[37] B.M. Craven, Acta Crystallogr. B35 (1979) 1123^1128.[38] J.A.W. Barnard, J.E. Lydon, Mol. Cryst. Liq. Cryst. 26

(1974) 285^296.[39] H.S. Shich, C.E. Nordman, Abst. Am. Cryst. Assoc.,

Summer Meeting at Michigan State University, 1977 p. 84.[40] C.H. Carlisle, D. Crowfoot, Proc. R. Soc. Lond. Ser. A 184

(1945) 64^83.[41] P. Sawzik, B.M. Craven, Acta Crystallogr. B35 (1979) 789^

791.[42] N.G. Guerina, B.M. Craven, J. Chem. Soc. Perkin II (1979)

1414^1419.[43] V. Pattabhi, B.M. Craven, J. Lipid Res. 20 (1979) 753^759.[44] B.M. Craven, G.T. DeTitta, J. Chem Soc. Perkin II (1976)

814^822.

BBAMEM 77830 12-5-00


crystallization of the isobutylphosphocholine^cholesterol ... · vladimir a. karasev a;*, vladimir...

Documents